Presentation 18: Problems other than AHPND in EMS ponds, including the microsporidian Enterocytozoon hepatopenaei (EHP) (Dr Timothy W. Flegel, Thailand)

FAO TCP/INT/3502 “Reducing and managing the risk of
Acute Hepatopancreatic Necrosis Disease (AHPND) of Cultured Shrimp”
Problems other than AHPND
in EMS ponds
Tim Flegel
Centex Shrimp, Mahidol University and National Center for
Genetic Engineering and Biotechnology (BIOTEC), Thailand
timothy.fle@mahidol.ac.th
6/24/201
5
International Technical Seminar/Workshop “EMS/AHPND: Government, Scientist and Farmer Responses”
22-24 June 2015, Tryp Hotel, Panama City 1

6/24/201
5
International Technical Seminar/Workshop “EMS/AHPND: Government, Scientist and Farmer Responses” 2
• In Thailand there is now a high prevalence of
shrimp showing:
– Gregarine-like entities in the hepatopancreas (ATM)
(Sriurairatana et al. 2014. PLoS ONE. 9, e99170)
– A microsporidian (E. hepatopenaei) (EHP)
(Tangprasittipap et al. 2013. BMC Vet Res. 9:139)
– A new nodavirus (CMNV) reported from China (Zhang et
al. 2014. J Gen Virol (In press)
• Their relationship to AHPND (if any) is currently
unknown but should be investigated
• All are highly prevalent, indicating poor
biosecurity in hatcheries and ponds
New problems accompanying AHPND

6/24/201
5
• This is an epidemiological-based study aimed at
identifying risk factors for AHPND
• Includes 200 randomly, pre-selected Thai shrimp
ponds
• Altogether 184 samples have been received so far
and 150 analyzed for histology (74% of 200)
• The prevalence of ATM is around 80%
• PCR used to test for:
– Viruses covert mortality nodavirus (CMNV), yellow head virus (YHV)
and white spot syndrome virus (WSSV)
– The microsporidian Enterocytozoon hepatopenaei (EHP)
• Results are summarized in the following table
Thai cohort study of 200 ponds

Results from the cohort study
4
AHPND
No AHPND
but
bacterial
lesions
Early
mortality
but No HP
lesions
Collapsed HP
epithelium
Normal HP
03, 07, 11, 12,
16, 20, 32,
34,40, 45, 47,
50, 52, 54, 55,
58, 73, 74, 82,
83, 84, 87,
90,91, 92, 93,
105, 110, 112,
116, 119, 122,
126, 131, 144,
150
04, 05, 21, 24,
44, 51, 69, 75,
79, 86, 88, 96,
99, 101, 117,
130, 133, 136,
139, 143
57, 70, 98, 100,
111
02, 15, 17, 25, 26,
29, 30, 37, 38, 46,
48, 49, 53, 64, 65,
76, 80, 81, 95, 97,
104, 109, 113, 114,
115, 123, 125, 127,
128, 134, 135
01, 06, 08, 09, 10, 13, 14,
18, 19, 22, 23, 27, 28, 31,
33, 35, 36, 39, 41, 42, 43,
56, 59, 60, 61, 62, 63, 66,
67, 68, 71, 72, 77, 78, 85,
89, 94, 102, 103, 106, 107,
108, 118, 120, 121, 124,
129, 132, 137, 138, 140,
141, 142, 145, 148, 149
36/148
(24%)
20/148
(14%)
5/148
(3%)
31/148
(21%)
56/148
(38%)
Infected with the microsproidian E. hepatopenaei (EHP) 72/148 = 49%
Mortality <35 days (study definition of EMS = 36/148 = 24%)
Infected with white spot syndrome virus (WSSV) = 8/148 = 5%
Covert mortality nodavirus (CMNV) = 64/148 = 43%
Specimens 146 and 147 un-readable (poor fixation)

6/24/201
5
Progress with the microsporidian
Enterocytozoon hepatopenaei
(EHP)

6/24/201
5
What is still unknown about EHP
• Are there life stages in other host species?
– These may be identified by PCR screening,
microscopy and in situ hybridization
• What are the modes of transmission in shrimp?
– These may be determined by co-habitation, feeding
and bath exposure to purified spores
• How can the spores be inactivated?
– This may be determined using viability assays and/or
an infection model
• Is therapeutic treatment possible?
– This may be determined using naturally infected
shrimp or using a infection model

6/24/201
5
Life stages and screening
• Our current nested-PCR method based on 16S
rRNA is OK for HP samples from infected shrimp
• Possibility for cross reactions with closely related
species (unknown?) in environmental samples
• We are cooperating with CEFAS (UK) to sequence
the genome from purified spore DNA
• This will allow us to choose a more specific marker
gene for screening
• So far, polychaetes, clams & snails PCR positive
• Must confirm infection by in situ hybridization
• Our new in situ LAMP method will help

Nested PCR for 16S rRNA of EHP
94C 94C
58C/64C
72C
72C
16C
35 cycles
5min
7min
20 sec
45sec/20sec
20 sec
779bp
176bp

6/24/201
5
Need for purified spores
• This was needed to carry out several tasks:
– Preparing DNA for genome sequencing
– Tests on spore inactivation
– Quantitative spore transmission tests
• An appropriate infection model is needed for use
of the purified spores

6/24/201
5
Modes of transmission
• Our student from India Paul Vinu Salachan has
done much of this work
• Transmission successful by feeding infected HP
tissue and by cohabitation
• So far, feeding of purified spores has not been
successful
• This raises many questions about the mode of
infection by cohabitation

6/24/201
5
Feeding trials with 2 g shrimp
Group Feed Dosage Description Replicates
Control
Commercial feed
pellet
10% BW/day NA 2 x 10
Spores
Commercial feed
pellet + 1.5 X 107
spores
10% BW/day for
12 days*
Top coated 2 x 10
HP tissue
Infected
Hepatopancreas
10% BW/day for 3
days#
Freshly
harvested
1 x 4**
Temperature set at 32C
After treatment feeds were commercial pellets to day 19
*6.25x104
spores/day/shrimp = 7.5x105
spores/shrimp total
** Repeat of Tangprasittipap et al. 2013. BMC Veterinary Research. 9, 139

Control HP fed
N P M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M
N P M 1 2 3 4 5 6 7 8 9 10
PCR confirmation of infection
Spore fed group
all negative

Confirmation in-situ Hybridization

N P M 1 2 3 4 5 6
Naïve shrimp outside
cage + infected shrimp
inside
Raised for 14 days, sacrificed
for histology and PCR
DNA
extraction &
PCR
All control shrimps were negative
Cohabitation trial

PCR results
N P M 1 2 3 4 5 6
176 bp
779 bp
All positive

Alternative infection models
• Cohabitation is the best infection model we have
so far (better than feeding HP tissue)
• Has disadvantages of needing infected shrimp, 2-
week test time, no pathogen quantification
• Warachin and Anuwat are testing cell culture
models using purified EHP spores
• Sf9 insect cells failed but C6/36 mosquito cells
show indications of early infection stages only
• Shrimp primary HP tissue cells still to be tested
• Such models would allow for rapid screening of
potentially protective treatments

6/24/201
5
Spore inactivation and therapy
• Paul failed with several reported methods of
testing microsporidian spore inactivation:
– By inhibition of apical filament release
– By using various normal and fluorescent vital dyes
• A coccidiostat has been tested with infected
shrimp but gave no infection decrease
• It would be better to test such compounds using
the cohabitation infection model

6/24/201
5
Gregarine-like entity (GLE)
(Now called aggregated transformed microvilli)
(ATM)
Sriurairatana et al. 2014. PLoS ONE. 9: e99170.

ATM
aggregation
steps in
H&E sections

Transformation & peeling process 2

Final aggregation and condensation

6/24/201
5
Distorted HP tubules
• Another anomaly of unknown cause.
• Grossly normal PL or young juveniles.
• Is this caused by a toxin?
• Is it associated with AHPNS?

Fresh mounts of HP tissue
normal

6/24/201
5
A word about two new viruses
from China

6/24/201
5
CMNV and a new type of YHV
• As I reported last time, we have found covert
mortality nodavirus (CMNV) common in Thailand
• However, our challenge tests have resulted in no
shrimp mortality or other signs of disease
• In situ hybridization tests give positive results only in
the nuclei of normal HP tubule epithelial cells
• It is possible that it can act together with other agents
for factors to cause disease
• The sequence for the partial polymerase gene of a
new YHV is available at GenBank KF278563
• We should probably prepare specific RT-PCR primers
and check our shrimp

The King’s project at Kungkabaen

Presentation 18: Problems other than AHPND in EMS ponds, including the microsporidian Enterocytozoon hepatopenaei (EHP) (Dr Timothy W. Flegel, Thailand)

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Presentation 18: Problems other than AHPND in EMS ponds, including the microsporidian Enterocytozoon hepatopenaei (EHP) (Dr Timothy W. Flegel, Thailand)

Similar to Presentation 18: Problems other than AHPND in EMS ponds, including the microsporidian Enterocytozoon hepatopenaei (EHP) (Dr Timothy W. Flegel, Thailand) (20)

More from ExternalEvents

More from ExternalEvents (20)

Recently uploaded

Recently uploaded (20)

Presentation 18: Problems other than AHPND in EMS ponds, including the microsporidian Enterocytozoon hepatopenaei (EHP) (Dr Timothy W. Flegel, Thailand)